1. Field of the Invention
The present invention relates generally to spectroscopy and microscopy. More particularly, embodiments of the present invention provide methods and systems for wide-field coherent anti-Stokes Raman scattering (CARS) microscopy using multiple nonlinear scattering channels to produce multiple, spatially coherent CARS beams from thin samples.
2. Description of Related Art
Optical microscopy has been an indispensable imaging tool in the life and materials sciences for over a century. Unlike electron microscopy, optical techniques offer the potential for studying living systems. Modern microscopes and sophisticated staining techniques routinely provide investigators with views of the workings of cells, both fixed and living. However, a number of researchers and funding organizations have underscored scientific challenges that often exceed the limits of traditional microscopy. For example, there are cellular processes in which fluorescent stains used to distinguish specific functional groups in a cell are either unavailable or undesirable. These extrinsic markers can disrupt normal biochemical processes. As such, imaging contrast based on intrinsic chemical signatures of the molecules under study would be a significant advantage. In addition, high speed or dynamic processes and enhanced spatial resolution are desirable in microscopy.
One approach to functional and chemically-resolved microscopy is to employ vibrational contrast imaging using, for example, Raman emission or infrared absorption. While they do not provide the detailed atomic location obtained in diffraction experiments, vibrational spectroscopies are powerful, for example in the study of biomacromolecules. Raman scattering is of particular interest because it can be excited by visible laser wavelengths, providing significantly greater spatial resolution than infrared microscopy. Vibrational spectra reveal a wealth of information concerning the chemistry, structure, conformation, and interactions of the molecules under study. For example, proteomic analyses, including post-translational modifications such as glycosylation and tyrosine phosphorylation have been measured using Raman scattering. ATP and GTP hydrolysis have been measured spectroscopically by monitoring changes in nucleoside triphosphohydrolase Raman bands.
A principal disadvantage of Raman spectromicroscopy is that Raman scattering is a weak process, typically 10-100 trillion times weaker than emission from efficient fluorophores. A number of groups have experimented with physical and chemical techniques to increase Raman scattering efficiency using, for example, electrodynamic enhancement produced by proximate metal surfaces. Nonlinear vibrational spectroscopies, most notably Coherent Anti-Stokes Raman Scattering (CARS), have been used for many years primarily in the study of combustion processes. Because CARS is generated coherently, the signal strength varies quadratically as the number of molecules excited increases. Signal strengths can be orders of magnitude higher in CARS than in conventional spontaneous Raman scattering.
CARS microscopy typically employs tight focusing of pump and Stokes beams and collinear illumination/collection geometries which facilitate laser wavelength tuning while maintaining the phase-matching condition. Spatial resolution of approximately 300 nm in the lateral direction and approximately 800 nm axially have been reported. Near-IR picosecond lasers are used to reduce autofluorescent background and provide good spectral resolution below 5 cm−1, suitable for many condensed systems. Because the anti-Stokes emission is generated coherently, CARS microscopy is significantly faster and more sensitive than conventional Raman microscopy. For example, multiplex CARS microscopy, in which spectrally broad anti-Stokes pulses are stimulated from the sample, has recently been demonstrated. The image acquisition times are roughly sixty times shorter than those in a conventional Raman microscope using identical average power levels and identical samples. Narrow-band CARS microscopy can be up to four orders of magnitude faster than spontaneous Raman microscopy. This also allows investigators to work with significantly lower average power delivered to the sample than would be required in spontaneous Raman, thus reducing potential photo- or thermal damage to fragile specimens.
Scanned microscopy is currently the dominant technique for CARS as well as 3D fluorescence imaging, in part because of the ease of image interpretation and the wide availability of commercial instruments for both linear and multiphoton fluorescence imaging. However, scanned microscopy does not produce sub-wavelength resolution.
Any shortcoming mentioned above is not intended to be exhaustive, but rather is among many that tends to impair the effectiveness of previously known imaging techniques using Raman spectromicroscopy; however, shortcomings mentioned here are sufficient to demonstrate that the methodologies appearing in the art have not been satisfactory and that a significant need exists for the techniques described and claimed in this disclosure.